The subject invention relates to frits configured for use in high pressure liquid chromatography (HPLC) chromatographic columns, and more particularly, to a frit capable of retaining sub-2.5 micrometer particles. The frits also can serve as in-line filters arranged upstream of chromatographic columns in HPLC systems. A high pressure liquid chromatography (HPLC) column is widely used for the separation and quantification of solutes in a liquid stream. A typical chromatographic system, as shown in FIG. 7, includes at least the following major components: a pump 70, an injector 72, a chromatographic column 74, a detector 76, and a computer 78 running software capable of data acquisition and processing. The pump 70 is used to propel a liquid stream through the injector, column, and detector. The injector 72 is operably connected to the pump 70 and permits the introduction of a small volume of a liquid sample into the liquid stream prior to its entering the column 74. Sample components are then separated as they migrate through the column by means of a variety of interactions between the solutes and the packing material contained therein. Upon exiting the column the individual components are detected by the detector 76, before being discarded. A signal from the detector 76 is then processed by a suitable computer software program in the computer 78 to provide a numerical value indicating the amount of solute detected.
Central to advances in the field of HPLC are advances in packing material technology and theory. The particle sizes of chromatographic packing materials used in HPLC columns have been decreasing over time. In the late 1960's, 30 micrometer packings were in use. By the early 1970's, packing technologies were developed to provide reproducible columns containing 10 micrometer packing materials. In the late 1970's, columns packed with 5 micrometer packings became commercially available. During the 1980's and 1990's, improvements in the purity and surface derivatizations of the packing materials further advanced the science. Presently, 3 micrometer packing materials are in common use. With the 1999 introduction of 2.5 micrometer packings by Waters Corporation of Milford, Mass., the trend clearly is toward packing materials with smaller particle sizes.
Sintered porous filters are widely used at the inlet and outlet of chromatographic columns for the retention of the particulate packing material in HPLC columns. Sintered filters are typically made by compacting particles having a controlled particle size distribution into a desired shape, and then sintering to form an interconnected network of pores within the filter. Filters commonly used for chromatographic purposes can be made from a variety of materials, such as stainless steel, titanium, polyetheretherketone (PEEK), or polyethylene. The majority of HPLC columns in use today are manufactured using 316 stainless steel filters, since this material provides a good balance of high strength, low cost, and corrosion resistance.
The grade, or nominal particle retention rating, of a chromatographic frit within an HPLC column is chosen on a case-by-case basis as a function of the particles to be contained within the column. Porous sintered stainless steel frits used in chromatographic columns containing 5 micron or 3 micron particles typically use 2.0 or 0.5 grade media frits, respectively. Media grades can be derived from a combination of air flow, porosity, and particle retention measurements, and do not necessarily equate to the actual pore size through the filter. Filters are available from several sources such as VICI (Valco Instrument Co.) of Houston, Tex.; Alltech Associates Inc. of Deerfield, Ill.; and Mott Corporation's Porous Metal Products of Farmington, Conn. Although such porous filters are capable of retaining particles as small as 2.5 micrometers in diameter under HPLC conditions, these filters have difficulty adequately retaining particles less than 2.5 microns in diameter.
The channels through conventional frits are significantly larger than the particles the frit is designed to retain. In use, the frits behave as depth filters, where retention is accomplished through particle-particle and particle-wall interactions that block the tortuous path of the channels. The particle retention efficiency of such filters varies with the flow rate, particle size, and concentration of the challenge fluid. Retention under a given set of conditions does not guarantee retention under various conditions encountered in HPLC.
Challenging a nominal 0.5 grade frit with solutions containing sub-2.5 micron packing material has been shown to produce a cloudy effluent downstream of the frit, which is evidence of particle breakthrough. FIG. 8 is an example of a 0.5 grade frit packed with 2.2 micron chromatographic packing material. The frit was inserted in an outlet fitting and placed in a chromatography column for about one hour prior to disassembly, at which time the outlet fitting and frit were removed from the column. FIG. 8 is a scanning electron micrograph of the downstream side of the frit, which indicates that the packing material migrated through the frit, as evidenced by the contamination of the downstream side of the frit with the packing material. In addition, during use, columns containing sub-2.5 micron packings configured with conventional frits produce very sharp intermittent spikes in the baseline of UV chromatograms, which is indicative of particle breakthrough. For example, FIG. 9 shows UV chromatograms of the elements of three (A-C) HPLC columns packed with 1.7 micron chromatographic packing materials, which included conventional 0.5 grade frits. The spikes in the chromatograms are a clear indication of migration of packing material through the conventional frits. Therefore, under the desired use conditions, complete particle retention is not achieved using conventional frits.
Adequate retention of the chromatographic packing material is imperative to the mechanical stability of the column and the integrity of the HPLC system. It is particularly important when separation conditions demand very high column efficiencies. In order to achieve high efficiency in a minimal amount of time, the smallest possible particle size packings are desirable. The HPLC system's extra column tubing volume must be minimized in order not to detract from the efficiency performance of the column. This requires the use of very small diameter connection tubing, which can be easily plugged by particles if they are not well retained within the HPLC column. Conventional frits do not adequately retain sub-2.5 micrometer packings.
Sintered porous metal filters capable of retaining small particulates are typically made by pressing or molding metal or metal alloy powders into a desired shape. The formed shape is then sintered at high temperatures to provide a consolidated porous object. These porous materials are manufactured for specific applications and have characteristics that are dependent on the size, shape, and type of powder, in addition to the compression and temperature used in the process. Presently, frits used in HPLC columns are produced using 45-100 micrometer irregularly shaped powders as starting materials. Sintering powders of sub-10 micrometer particulate size and of a spherical shape is difficult. The difficulty in handling compressed forms made from <10 micrometer spherical particle size powders is due to poor mechanical strength of the “green” form prior to sintering. The poor mechanical strength makes the green forms too unstable to withstand the handling and transfer required in the sintering process. In addition, green forms produced by compacting <10 micron spherical powders tend to shrink excessively upon sintering, resulting in the formation of cracks and channels in the final frit structure.
Retention of sub-2.5 micrometer particles requires a finer pore structure than exists in conventional HPLC frits. A solution to this problem can be to compress further the existing frit media to further close off the pores and narrow the channel openings. However, this solution has the disadvantage of decreasing porosity and hence reducing permeability of the frit. Alternatively, smaller particle size powders can be used, but such powders suffer from poor “green” strength and excessive shrinkage during sintering. A number of patents propose ways to deal with this problem, frequently encountered in the filtration of gases for the semiconductor industry. In all cases a mechanically stable support is used to provide the needed strength either by layering on (U.S. Pat. Nos. 5,456,740; 4,746,341; 4,976,760; 4,039,703; and 5,925,156) or filling in the support (U.S. Pat. Nos. 5,114,447; 4,613,369; 4,888,114; and 6,080,219).
The subject invention overcomes the problems associated with conventional frits by providing a frit capable of retaining sub-2.5 micrometer packing materials, an HPLC system incorporating the frit, and a method of retaining sub-2.5 micrometer packing materials in HPLC columns.